Rapamycin-Induced Feedback Activation of eIF4E-EIF4A Dependent mRNA Translation in Pancreatic Cancer

Simple Summary Pancreatic cancer is aggressive cancer with a low survival rate due to the lack of detection, effective treatment, and development of therapeutic resistance. New treatments and mechanistic details of therapeutic resistance are urgently needed. In this study, we explored the effect of inhibiting protein synthesis and its role in inducing feedback mechanisms that may impact the therapeutic response. We show that Rapamycin (sirolimus) treatment inhibited the synthesis of proteins required for cancer cell growth. Interestingly, rapamycin treatment induced the synthesis of proteins that lead to reactivation of the key kinases, and this limited the anti-tumor effect of rapamycin. We further show that the combination of rapamycin with the small molecule inhibitor CR-1-31-B increases the efficacy of rapamycin. Our study establishes the feedback mechanism induced by rapamycin and new therapeutic combinations that can be further developed as therapeutics for pancreatic cancer. Abstract Pancreatic cancer cells adapt molecular mechanisms to activate the protein synthesis to support tumor growth. This study reports the mTOR inhibitor rapamycin’s specific and genome-wide effect on mRNA translation. Using ribosome footprinting in pancreatic cancer cells that lack the expression of 4EBP1, we establish the effect of mTOR-S6-dependent mRNAs translation. Rapamycin inhibits the translation of a subset of mRNAs including p70-S6K and proteins involved in the cell cycle and cancer cell growth. In addition, we identify translation programs that are activated following mTOR inhibition. Interestingly, rapamycin treatment results in the translational activation of kinases that are involved in mTOR signaling such as p90-RSK1. We further show that phospho-AKT1 and phospho-eIF4E are upregulated following mTOR inhibition suggesting a feedback activation of translation by rapamycin. Next, targeting eIF4E and eIF4A-dependent translation by using specific eIF4A inhibitors in combination with rapamycin shows significant growth inhibition in pancreatic cancer cells. In short, we establish the specific effect of mTOR-S6 on translation in cells lacking 4EBP1 and show that mTOR inhibition leads to feedback activation of translation via AKT-RSK1-eIF4E signals. Therefore, targeting translation downstream of mTOR presents a more efficient therapeutic strategy in pancreatic cancer.

[1]  A. Viale,et al.  Frequent 4EBP1 Amplification Induces Synthetic Dependence on FGFR Signaling in Cancer , 2022, Cancers.

[2]  C. Maracci,et al.  Targeting mTOR and eIF4E: a feasible scenario in ovarian cancer therapy , 2021, Cancer drug resistance.

[3]  Heshui Wu,et al.  Everolimus regulates the activity of gemcitabine-resistant pancreatic cancer cells by targeting the Warburg effect via PI3K/AKT/mTOR signaling , 2021, Molecular medicine.

[4]  Jing Tang,et al.  DrugComb update: a more comprehensive drug sensitivity data repository and analysis portal , 2021, bioRxiv.

[5]  Stefan G. Stark,et al.  Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules , 2021, Cancer Research.

[6]  OUP accepted manuscript , 2021, Nucleic Acids Research.

[7]  J. D'haese,et al.  mTORC1 and mTORC2 converge on the Arp2/3 complex to promote KrasG12D-induced acinar-to-ductal metaplasia and early pancreatic carcinogenesis. , 2020, Gastroenterology.

[8]  A. Basu,et al.  Distinct Roles of mTOR Targets S6K1 and S6K2 in Breast Cancer , 2020, International journal of molecular sciences.

[9]  E. Jacinto,et al.  Targeting mTOR and Metabolism in Cancer: Lessons and Innovations , 2019, Cells.

[10]  S. Ethier,et al.  Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer , 2019, BMC Cancer.

[11]  Dirk Mossmann,et al.  mTOR signalling and cellular metabolism are mutual determinants in cancer , 2018, Nature Reviews Cancer.

[12]  Philippe P Roux,et al.  Signaling Pathways Involved in the Regulation of mRNA Translation , 2018, Molecular and Cellular Biology.

[13]  M. Candeias,et al.  Cap-independent translation ensures mTOR expression and function upon protein synthesis inhibition , 2017, RNA.

[14]  David M. Sabatini,et al.  mTOR Signaling in Growth, Metabolism, and Disease , 2017, Cell.

[15]  Gunnar Rätsch,et al.  RiboDiff: detecting changes of mRNA translation efficiency from ribosome footprints , 2015, bioRxiv.

[16]  N. Sonenberg,et al.  Signalling to eIF4E in cancer , 2015, Biochemical Society transactions.

[17]  Konstantinos J. Mavrakis,et al.  RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer , 2014, Nature.

[18]  J. Pelletier,et al.  Pancreatic tumours escape from translational control through 4E-BP1 loss , 2014, Oncogene.

[19]  Wei Shi,et al.  featureCounts: an efficient general purpose program for assigning sequence reads to genomic features , 2013, Bioinform..

[20]  N. Sonenberg,et al.  mTORC1 controls mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation. , 2013, Cell metabolism.

[21]  K. Shokat,et al.  Myc and mTOR converge on a common node in protein synthesis control that confers synthetic lethality in Myc-driven cancers , 2013, Proceedings of the National Academy of Sciences.

[22]  David G Hendrickson,et al.  Differential analysis of gene regulation at transcript resolution with RNA-seq , 2012, Nature Biotechnology.

[23]  Anna M. McGeachy,et al.  The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments , 2012, Nature Protocols.

[24]  Gerald C. Chu,et al.  Oncogenic Kras Maintains Pancreatic Tumors through Regulation of Anabolic Glucose Metabolism , 2012, Cell.

[25]  D. Sabatini,et al.  A unifying model for mTORC1-mediated regulation of mRNA translation , 2012, Nature.

[26]  Nicholas T. Ingolia,et al.  The translational landscape of mTOR signalling steers cancer initiation and metastasis , 2012, Nature.

[27]  D. Bar-Sagi,et al.  RAS oncogenes: weaving a tumorigenic web , 2011, Nature Reviews Cancer.

[28]  E. Peng,et al.  Rapamycin regulates Akt and ERK phosphorylation through mTORC1 and mTORC2 signaling pathways , 2010, Molecular carcinogenesis.

[29]  Cole Trapnell,et al.  Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. , 2010, Nature biotechnology.

[30]  D. Sabatini,et al.  An ATP-competitive Mammalian Target of Rapamycin Inhibitor Reveals Rapamycin-resistant Functions of mTORC1* , 2009, Journal of Biological Chemistry.

[31]  Sang Gyun Kim,et al.  Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation , 2008, Proceedings of the National Academy of Sciences.

[32]  S. Lowe,et al.  Dissecting eIF4E action in tumorigenesis. , 2007, Genes & development.

[33]  M. Sandri,et al.  S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover. , 2007, American journal of physiology. Cell physiology.

[34]  L. Helman,et al.  Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism , 2007, Oncogene.

[35]  Steven P. Gygi,et al.  mTOR and S6K1 Mediate Assembly of the Translation Preinitiation Complex through Dynamic Protein Interchange and Ordered Phosphorylation Events , 2005, Cell.

[36]  F. Khuri,et al.  Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. , 2005, Cancer research.

[37]  J. C. Schmitz,et al.  Translational Regulation as a Novel Mechanism for the Development of Cellular Drug Resistance , 2004, Cancer and Metastasis Reviews.

[38]  C. Proud,et al.  Regulation of elongation factor 2 kinase by p90RSK1 and p70 S6 kinase , 2001, The EMBO journal.

[39]  S. Gygi,et al.  Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism. , 1999, Genes & development.

[40]  E. Gelfand,et al.  Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  S. Sehgal,et al.  Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization. , 1975, The Journal of antibiotics.

[42]  S. Sehgal,et al.  Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. , 1975, The Journal of antibiotics.